The initiating event in the pathogenesis of atherosclerosis and restenosis following angioplasty is injury to cells in the artery wall1. Injury or stress stimulates signalling and transcriptional pathways in vascular smooth muscle cells, stimulating their migration and proliferation and the eventual formation of a neointima. Smooth muscle cell proliferation is a key feature of neointima formation, atherosclerosis, restenosis and graft failure.
c-Jun, a prototypical member of the basic region-leucine zipper protein family, is transiently induced following arterial injury in animal models2,3. c-Jun forms both homodimers and heterodimers with other bZIP proteins to form the AP-1 transcription factor. While investigations over the last decade have linked AP-1 with proliferation, tumorigenesis and apoptosis, AP-1 has also been implicated in tumor suppression and cell differentiation4. Thus, gene-targeting strategies that down-regulate c-Jun expression do not necessarily inhibit cell proliferation.
Kanatani et al, (1996)5 have shown that antisense oligonucleotides targeting c-Jun dose-dependently reduce the growth-inhibitory effect of dexamethasone and TGFβ. Recent reports indicate that c-Jun NH2-terminal kinase/stress activated protein kinase (JNK), an upstream activator of c-Jun and numerous other transcription factors, is expressed by SMCs in human and rabbit atherosclerotic plaques6,7 and that dominant negative JNK inhibits neointima formation after balloon injury8. c-Jun, however, has not been localised in human atherosclerotic lesions, nor has it been shown to play a functional role in arterial repair after injury.
It is clear, however, that the finding that c-Jun, or any other given gene, is inducibly expressed in the artery wall following balloon angioplasty does not necessarily translate to it playing a positive regulatory role in transcription, proliferation or neointima formation. For example, it has been shown that three transcriptional repressors (NAB2, GCF2, and YY1) are activated in vascular smooth muscle cells by mechanical injury in vitro, as well as in the rat artery wall. NAB2 directly binds the zinc finger transcription factor Egr-1 and represses Egr-1-mediated transcription9. GCF2 is a potent repressor of the expression of PDGF-A, a well-established mitogen for vascular smooth muscle cells, and inhibits smooth muscle cell proliferation10. Similarly, YY1 overexpression blocks smooth muscle cell growth without affecting endothelial cell proliferation11.
c-Jun can repress, as well as activate transcription. c-Jun binds the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity12. c-Jun also blocks transforming growth factor beta-mediated transcription by repressing the transcriptional activity of Smad313.
c-Jun can inhibit, as well as stimulate proliferation. Using antisense oligonucleotides to c-Jun, Kanatani and colleagues demonstrated that inhibition of human monocytoid leukemia cell growth by TGF-beta and dexamethasone is mediated by enhanced c-Jun expression5.
c-Jun, however, has not been directly linked to the complex process of angiogenesis, which underlies many common human diseases including solid tumor growth and corneal disease. Angiogenesis is a complex multi-step process involving proteolytic degradation of the basement membrane and surrounding extracellular matrix, microvascular endothelial cell proliferation, migration, tube formation and structural re-organisation14.
DNAzymes
In human gene therapy, antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable. The anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, an mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression by mechanisms including nucleolytic degradation or steric blockade of the translational machinery.
Anti-sense technology suffers from certain drawbacks. Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex. This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component. Here, the DNA anti-sense molecule serves in a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme. This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNA's. Anti-sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.
As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature15-21. Thus, unlike a conventional anti-sense molecule, a catalytic nucleic acid molecule functions by actually cleaving its target mRNA molecule instead of merely binding to it. Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements. The target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage.
Catalytic RNA molecules (“ribozymes”) are well documented15,22,23, and have been shown to be capable of cleaving both RNA15 and DNA20 molecules. Indeed, the development of in vitro selection and evolution techniques has made it possible to obtain novel ribozymes against a known substrate, using either random variants of a known ribozyme or random-sequence RNA as a starting point16,24,25.
Ribozymes, however, are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications.
Recently, a new class of catalytic molecules called “DNAzymes” was created26,27. DNAzymes are single-stranded, and cleave both RNA16,27 and DNA21. A general model for the DNAzyme has been proposed, and is known as the “10-23” model. DNAzymes following the “10-23” model, also referred to simply as “10-23 DNAzymes”, have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of variable deoxyribonucleotide arm length. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions27.
DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule is not predictable. This unpredictability is due, in part, to two factors. First, certain mRNA secondary structures can impede a DNAzyme's ability to bind to and cleave its target mRNA. Second, the uptake of a DNAzyme by cells expressing the target mRNA may not be efficient enough to permit therapeutically meaningful results.
Investigation of the precise regulatory role of c-Jun in the injured artery wall and indeed, in other disease settings such as angiogenesis, has been hampered by the lack of a specific pharmacological inhibitor. DNAzymes represent a new class of gene targeting agent with specificity conferred by the sequence of nucleotides in the two arms flanking a catalytic core27, with advantages over ribozymes of substrate specificity and stability27,28. To date, neither c-Jun nor indeed any other Jun family member has been targeted using catalytic nucleic acid strategies.